Abstract Background: Urinary bladder cancer is an important health concern in developed countries, particularly for men, among whom incidence is 3-5 times greater than in women. Vitamin D is thought to protect against cancer at many sites. However, to our knowledge, no studies have examined the association between serum vitamin D and bladder cancer. Methods: Within the ATBC Study, a randomized controlled trial conducted to determine the effects of α-tocopherol and β-carotene supplements on cancer incidence, 250 bladder cancer cases were randomly sampled by month of blood collection such that there were 25 cases included from each month with 25 cases total from June - August because fewer blood collections were conducted during the summer months. Controls were sampled from ATBC study participants who were alive and cancer free at the time the case was diagnosed and were matched 1:1 with cases on age at randomization (± 1 year) and date of blood collection (± 30 days). Conditional logistic regression was used to estimate odds ratios and 95% confidence intervals of bladder cancer by a priori categories of baseline serum 25-hydroxy vitamin D (<25, 25 - <37.5, 37.5 - <50, ≥ 50 nmol/L). Results: After multivariable adjustment, lower serum vitamin D was associated with a statistically significantly increased risk of bladder cancer (OR, 95% CI vs. ≥ 50 nmol/L; 37.5 - <50 nmol/L: 2.04, 1.13 − 3.70; 25 - <37.5 nmol/L: 1.90, 1.07 − 3.35; <25 nmol/L: 1.83, 1.06 − 3.14; p-trend = 0.03), although the risk did not appear to increase with progressively lower vitamin D levels. Rather, men in any category of serum vitamin D below 50 nmol/L were at approximately two-fold risk compared to men with serum vitamin D ≥50 nmol/L (OR, 95% CI: 1.91, 1.20 − 3.04; p value = 0.006). Although the results were not statistically significant, there was a suggestion that the vitamin D - bladder cancer association was stronger among men who smoked less, were not assigned to the α-tocopherol intervention arm, or whose blood samples were obtained during the sunnier months. Conclusion: In this population of male smokers, lower vitamin D was associated with an increased risk of bladder cancer. Future studies should examine this association in other populations, especially women and non-smokers. Replication in a larger sample may clarify possible subtle effect modification by smoking, season, and intake of other nutrients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2799.